香叶酸(GGA),作为第二原发性肝癌的预防剂,据报道,在人肝癌衍生细胞中通过甲羟戊酸途径进行生物合成。最近,我们发现单胺氧化酶B(MAOB)催化香叶基香叶醇(GGOH)的氧化产生香叶基香叶醇(GGal),肝癌细胞中内源性GGA的直接前体,使用tranylcypromine,MAOs的抑制剂,并通过MAOBsiRNA敲除。然而,在使用CRISPR-Cas9系统建立的MAOB敲除(KO)细胞中,内源性GGA水平出乎意料地没有变化,提示一些其他潜在的代谢途径维持MAOB-KO细胞中的内源性GGA水平。这里,我们研究了在Hep3B/MAOB-KO细胞中氧化GGOH的潜在酶。首先,广泛特异性细胞色素P450酶抑制剂以剂量依赖性方式降低了Hep3B/MAOB-KO细胞中内源性GGA的含量.第二,在先前研究中被认为参与异戊二烯醇和/或视黄醇氧化的细胞色素P450超家族的八个成员中,只有CYP3A4基因在Hep3B/MAOB-KO细胞中显著上调其细胞mRNA水平。第三,一种市售的重组人CYP3A4酶能够将GGOH氧化为GGal,第四,siRNA对CYP3A4的敲除显著降低了Hep3B/MAOB-KO细胞中内源性GGA的含量。这些结果表明,当人肝癌来源的细胞系Hep3B中的肝MAOB缺失时,CYP3A4可以作为GGOH的替代氧化酶,并且内源性GGA水平由多种酶维持。
Geranylgeranoic acid (GGA), developed as a preventive agent against second primary hepatoma, has been reported to be biosynthesized via the mevalonate pathway in human hepatoma-derived cells. Recently, we found that monoamine oxidase B (MAOB) catalyzed the oxidation of geranylgeraniol (GGOH) to produce geranylgeranial (GGal), a direct precursor of endogenous GGA in hepatoma cells, using tranylcypromine, an inhibitor of MAOs, and knockdown by MAOB siRNA. However, endogenous GGA level was unexpectedly unchanged in MAOB-knockout (KO) cells established using the CRISPR-Cas9 system, suggesting that some other latent metabolic pathways maintain endogenous GGA levels in the MAOB-KO cells. Here, we investigated the putative latent enzymes that oxidize GGOH in Hep3B/MAOB-KO cells. First, the broad-specific cytochrome P450 enzyme inhibitors decreased the amount of endogenous GGA in Hep3B/MAOB-KO cells in a dose-dependent manner. Second, among the eight members of cytochrome P450 superfamily that have been suggested to be involved in the oxidation of isoprenols and/or retinol in previous studies, only the CYP3A4 gene significantly upregulated its cellular mRNA level in Hep3B/MAOB-KO cells. Third, a commercially available recombinant human CYP3A4 enzyme was able to oxidize GGOH to GGal, and fourth, the knockdown of CYP3A4 by siRNA significantly reduced the amount of endogenous GGA in Hep3B/MAOB-KO cells. These results indicate that CYP3A4 can act as an alternative oxidase for GGOH when hepatic MAOB is deleted in the human hepatoma-derived cell line Hep3B, and that endogenous GGA levels are maintained by a multitude of enzymes.